The Expression Transcriptional Regulation Of Visfatin Gene In Adipocyte Of Goat

Visfatin is a cytokine which is produced secreted by adipose tissue and involve in the regulation of immunoreactions,inflammation and lipid metabolism through the endocrine or paracrine.Visfatin plays an important role in the regulation of lipid metabolism in adipose tissue.However,the information regarding visfatin and its transcription regulation in goats is limited.PPAR? is a key gene in adipocyte differentiation and activates adipokine genes.Rosiglitazone is a PPAR? agonist and can promote the expression of PPARy to increase the expression of lipogenesis-related genes.Therefore,investigation of the relationship between rosiglitazone and visfatin will help us to better understand the function of PPARy in lipid metabolism in Tibetan goats.In this study,we cloned the visfatin(visfatin)genes from non-pregnant female Tibetan goat adipose tissue,and analyzed the mRNA levels in tissue and adipocyte by qPCR.In addition,oil red O staining was used to observe the changes of lipid droplets during adipocyte differentiation and studied the effect of rosiglitazone on adipocyte differentiation.Besides,the promoter vector of visfatin gene was constructed,and the transcriptional regulation of visfatin gene was analyzed by the dual luciferase reporter gene detection system.The main results are as follows:1.The full-length coding sequences of visfatin were obtained and aligned the cDNA sequences with the goat genome database,chromosome locations were identified at chromosomes 4.The cDNA sequences of visfatin encode predicted proteinswith 491 amino acids.Remarkably,these domains are well conserved between goat,sheep,bovine,mouse and human,and the similarity is 99%,98%and 98%,respectively,which suggest that they are also evolutionarily conserved.Conserved domain prediction showed that the structure of visfatin belongs to the PRTase-type ? super family and is a nicotinate phosphoribosyltransferase.2.Visfatin were predominantly expressed in the kidney,and then is the brain and subcutaneous adipose tissue,which is relatively low in other tissues.3.The qPCR and oil red O stainingresults indicated that the accumulation of lipid droplets was increased during goat adipocyte differentiation,and the expression patterns of visfatin and PPAR increased and then decreased.The number of lipid droplets was increased,and the volume of lipid droplets was also increase during differentiation.visfatin was expressed at day 0 and day 2 at a relatively low level,significantly(p<0.01)increased at day 4,and peaked at day 6(p<0.01),whereas the expression level decreased from the day 6 to day 10.PPAR? was expressed at day 0 and day 2 at a relatively low level,significantly(p<0.01)increased to a peak at day 4,and then significantly(p<0.05)decreased at the day 6.4.In addition,the effect of PPAR? was observed by treated with rosiglitazone on differentiation of goat adipocytes.It was found that rosiglitazone significantly promoted the differentiation of goat f adipocytes and significantly influenced on the expression of visfatin and PPAR?.However,rosiglitazone significantly increased visfatin mRNA expression at day 2(p<0.01)but significantly decreased visfatin mRNA expression at day 4(p<0.05),day 6(p<0.01)and day 10(p<0.01).Rosiglitazone can promote the expression of PPAR?,with significant difference(p<0.01)from day 2 to day 6,and not significant at other stages.5.GSK3? significantly affected the expression of visfatin gene during the proliferation and differentiation stage.Overexpression of GSK3? was significantly reduced the expression of visfatin gene(p<0.05),whereas the mRNA level of visfatin gene significantly increased after SB216763 treatment(p<0.05).6.The activity of visfatin promoter was detected by the dual-luciferase reporter system,and the results showed that the promoter activity of-735/+34 was significantly higher than-1624/+34 and-486/+34.The analysis of visfatin gene promoter sequence showed that there was containing a PPAR? binding site in the-735/+34 segment.After the mutation of the binding site,the promoter activity was significantly reduced(p<0.01).The results of rosiglitazone treatment showed that the promoter activity of the visfatin gene-735/+34 was significantly inhibited(p<0.01),while the promoter fragment activity was not affected on PPAR? binding site mutation.Rosiglitazone had significantly inhibited the mRNA level of visfatin during the proliferation and differentiation stage(p<0.05),indicating that PPAR? could inhibit the expression of visfatin.7.Overexpression of GSK3? significantly increased the expression of PPARy during adipocytes proliferation and differentiation(p<0.05),and SB216763 significantly inhibited the expression of PPAR? during proliferation and differentiation stage(p<0.05).8.The binding capacity of PPAR? with promoter sequences was analyzed by ChIP assay.PPAR? was capable of binding to the visfatin promoter,and rosiglitazone decreased the binding capacity of PPAR? to the visfatin promoter.In addition,GSK3?overexpression was also decreased the binding capacity of PPAR? to the visfatin promoter.However,the binding capacity was increased by treated with SB216763.The result was determined that GSK3? regulated the binding capacity of PPAR? to the visfatin promoter.